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Diagnosis of malaria involves identification of malaria parasite
or its antigens/products in the blood of the patient. Although this seems simple, the
efficacy of the diagnosis is subject to many factors. The different forms of the four
malaria species; the different stages of erythrocytic schizogony; the endemicity of
different species; the population movements; the inter-relation between the levels of
transmission, immunity, parasitemia, and the symptoms; the problems of recurrent malaria,
drug resistance, persisting viable or non-viable parasitemia, and sequestration of the
parasites in the deeper tissues; and the use of chemoprophylaxis or even presumptive
treatment on the basis of clinical diagnosis can all have a bearing on the identification
and interpretation of malaria parasitemia on a diagnostic test.
The diagnosis of malaria is confirmed by blood
tests and can be divided into microscopic and non-microscopic tests.
Microscopic Tests
For nearly a hundred years, the
direct microscopic visualization of the parasite on the thick and/or thin blood smears has
been the accepted method for the diagnosis of malaria in most settings, from the clinical
laboratory to the field surveys. The careful examination of a well-prepared and
well-stained blood film currently remains the "gold standard" for malaria
diagnosis.
The microscopic tests involve staining
and direct visualization of the parasite under the microscope.
1. Peripheral smear study
2. Quantitative Buffy Coat (QBC) test
Peripheral
smear study for malarial parasites - The MP test
Light microscopy of thick and thin stained blood
smears remains the standard method for diagnosing malaria.
It involves collection of a blood smear, its staining with Romanowsky stains and examination of the Red Blood Cells
for intracellular malarial parasites. Thick smears are 20–40 times more sensitive than thin smears for screening of
Plasmodium parasites, with a detection limit of 10–50 trophozoites/μl. Thin smears allow one to identify malaria
species (including the diagnosis of mixed infections), quantify parasitemia, and assess for the presence of schizonts,
gametocytes, and malarial pigment in neutrophils and monocytes. The diagnostic accuracy relies on the quality of the
blood smear and experience of laboratory personnel.
Before reporting a negative result, at least 200 oil
immersion visual fields at a magnification of 1000× should be examined on both thick and thin smears, which has a
sensitivity of 90%. The level of parasitemia may be expressed either as a percentage of parasitized erythrocytes or
as the number of parasites per microliter of blood. In nonfalciparum malaria, parasitemia rarely exceeds 2%, whereas
it can be considerably higher (>50%) in falciparum malaria. In nonimmune individuals, hyperparasitemia (>5% parasitemia
or >250 000 parasites/μl) is generally associated with severe disease.
In falciparum malaria, parasitized erythrocytes may be
sequestered in tissue capillaries resulting in a falsely low parasite count in the peripheral blood ('visible' parasitemia).
In such instances, the developmental stages of the parasite seen on blood smear may help to assess disease severity better
than parasite count alone. The presence of more mature parasite forms (>20% of parasites as late trophozoites and schizonts)
and of more than 5% of neutrophils containing malarial pigment indicates more advanced disease and a worse prognosis. One
negative blood smear makes the diagnosis of malaria very unlikely (especially the severe form); however, smears should be
repeated every 6–12 hours for 48 hours if malaria is still suspected.[1-5]
The smear can be prepared
from blood collected by venipuncture, finger prick and ear lobe stab. In obstetric
practice, cord blood and placental impression smears can be used. In fatal cases,
post-mortem smears of cerebral grey matter obtained by needle necropsy through the foramen
magnum, superior orbital fissure, ethmoid sinus via the nose or through fontanelle in
young children can be used.
Sometimes no parasites
can be found in peripheral blood smears from patients with malaria, even in severe
infections. This may be explained by partial antimalarial treatment or by sequestration of
parasitised cells in deep vascular beds. In these cases, parasites, or malarial pigment
may be found in the bone marrow aspirates. Presence of malarial pigment in circulating
neutrophils and monocytes may also suggest the possibility of malaria.
Staining
methods - See details
Quantitative Buffy Coat (QBC) test
Non-Microscopic Tests
Several attempts have been made to
take the malaria diagnosis out of the realm of the microscope and the microscopist.
Important advances have been made in diagnostic testing, including
fluorescence microscopy of parasite nuclei stained with acridine
orange, rapid dipstick
immunoassay, and Polymerase
Chain Reaction assays.[1] These
tests involve identification of the parasitic antigen or the antiplasmodial antibodies or
the parasitic metabolic products. Nucleic acid probes and immunofluorescence for the
detection of Plasmodia within the erythrocytes; gel diffusion,
counter-immunoelectrophoresis, radio immunoassay, and enzyme immunoassay for malaria
antigens in the body fluids; and hemagglutination test, indirect immunofluorescence,
enzyme immunoassay, immunochromatography, and Western blotting for anti-plasmodial
antibodies in the serum have all been developed. These tests have found some limited
applications in research, retrograde confirmation of malaria, investigation of cryptic
malaria, transfusion blood screening, and investigation of transfusion acquired
infections.
Rapid
Diagnostic Tests (RDTs)
detect species-specific circulating parasite antigens targeting either
the histidine-rich protein-2 of P. falciparum or a parasite-specific lactate dehydrogenase.
Although the dipstick tests may enhance diagnostic speed, microscopic examination remains mandatory in patients with
suspected malaria, because occasionally these dipstick tests are negative in patients with high parasitemia, and
their sensitivity below 100 parasites/μl is low.
Tests based on polymerase chain reaction for species-specific
Plasmodium genome are more sensitive and specific than are other tests, detecting as few as 10 parasites/μl blood. Antibody detection has no value in the diagnosis of acute malaria. It is mainly used for epidemiologic studies.[6-8]
Therefore, the simplest and surest test is the
time-honoured peripheral smear study for malarial parasites. None of the other newer tests
have surpassed the 'gold standard' peripheral smear study.
Remember this:
-
Ask for MP test in all cases of fever and
related symptoms and also whenever there is high level of suspicion.
-
MP test can be done at
any time. Do not wait for typical symptoms and signs or for chills.
-
A negative test DOES
NOT rule out malaria. Repeated tests may have to be done in all doubtful cases. Duration
of the illness, level of parasitemia, expertise of the technician and the method of
examination may all have a bearing on the result of the M.P. test.
Missed parasites -
SEE Misreport
References:
- Andrej Trampuz,
Matjaz Jereb, Igor Muzlovic, Rajesh M Prabhu. Clinical review:
Severe malaria. Critical Care 2003;7:315-323 Available at
http://ccforum.com/content/7/4/315
- Moody AH, Chiodini PL.
Methods for the detection of blood parasites. Clin Lab Haematol 2000;22:189-201.
- Torres JR. Malaria and babesiosis. Therapy of Infectious Diseases (Edited by: Baddour LM,
Gorbach SL). Philadelphia, PA: Saunders 2003;597-613.
- Nguyen PH, Day N, Pram TD,
Ferguson DJ, White NJ. Intraleucocytic malaria pigment and prognosis in
severe malaria. Trans R Soc Trop Med Hyg 1995;89:200-204.
- White NJ. Malaria.
Manson's Tropical Diseases (Edited by: Cook GC, Zumla AI, Weir J).
Philadelphia, PA: WB Saunders 2003;1205-1295
- Lee SH, Kara UA, Koay E, Lee MA, Lam S, Teo D.
New strategies for the diagnosis and screening of malaria. Int J Hematol 2002;76(suppl 1):291-293.
- Moody A. Rapid diagnostic tests for malaria parasites.
Clin Microbiol Rev 2002;15:66-78.
- Hanscheid T, Grobusch MP: How useful is PCR in the diagnosis of malaria?
Trends Parasitol 2002;18:395-398
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